Development of new multiepitope DNA vaccines for active immunotherapy against neuroblastoma combined with immune checkpoint blockade

神经母细胞瘤（NB）是一种具有挑战性的儿童癌症，预后差。最近，GD 2导向的免疫疗法将高风险NB患者的生存率提高了15%。与大多数被动免疫疗法一样，这种策略不会诱导持久的免疫应答，因此需要重复应用Ab。本课题旨在构建一种新的抗NB多表位DNA疫苗。我们以前成功地产生了双顺反子IL-15为基础的DNA疫苗对肿瘤相关抗原（TAA）酪氨酸羟化酶（TH）在NB中过表达。在我们的同基因小鼠模型中对该疫苗的评估清楚地显示，与不含IL-15的对照TH疫苗相比，IL-15的抗肿瘤功效增加。我们在DNA疫苗领域的专业知识和新知识将用于产生更有效的新一代多表位DNA疫苗。为了增加免疫原性，除TH外，还将其他NB特异性TAA（ALK、MYCN、生存素）整合到疫苗中。这允许即使在肿瘤细胞损失一些TAA后也具有抗肿瘤作用。为了诱导持久效应，将进行T辅助细胞的额外激活，刺激抗原呈递细胞（APC），从而激活肿瘤特异性细胞毒性T细胞（CTL）。为此，将编码通用T辅助细胞表位的DNA序列整合到疫苗中。此外，为了将TAA-和T辅助表位分别有效地装载到MHC-I和MHC-II分子上，溶酶体和蛋白酶体抗原呈递途径都将由具有增加的稳定性的突变泛素的DNA疫苗依赖性合成（聚泛素化）诱导。疫苗介导的IL-15合成将实现对APC的额外刺激。在生成和表征之后，将在我们的临床前NB模型中评估新DNA疫苗的抗肿瘤功效。为此，将利用新的基于非病毒脂质体的纳米颗粒疫苗接种策略。为了克服肿瘤介导的免疫应答抑制，将进行已显示在癌症中起重要作用的两个免疫检查点（PD-L1 + TIGIT）的额外阻断。总之，我们的目标是产生针对不同NB特异性TAA的新一代基于精氨酸的DNA疫苗，其将与免疫检查点阻断组合进行测试以诱导针对NB的持久免疫应答。

Neuroblastoma (NB) is a still challenging cancer of childhood with poor prognosis. Recently, GD2-directed immunotherapies improved survival of high-risk NB patients by 15%. As with most passive immunotherapies, such a strategy does not induce a long-lasting immune response and repeated Ab applications are necessary. This obstacle can be avoided by DNA vaccination.In the present project, we aim to generate a new mutiepitope DNA vaccine against NB. We previously successfully generated a bicistronic IL-15-based DNA vaccine against the tumor-associated antigen (TAA) tyrosine hydroxylase (TH) overexpressed in NB. Evaluation of this vaccine in our syngeneic mouse model clearly showed increased antitumor efficacy by IL-15 compared to the IL-15-free control TH vaccine. Our expertise and new knowledge in the field of DNA vaccination will be used for the generation of a more effective new generation multiepitope DNA vaccine against NB. To increase immunogenicity, further NB-specific TAAs (ALK, MYCN, survivin) will be integrated into the vaccine additionally to TH. This allows antitumor effects even after loss of some TAAs by tumor cells. To induce long-lasting effects, additional activation of T helper cells stimulating antigen presenting cells (APCs) and thereby activating tumor-specific cytotoxic T cells (CTLs) will be performed. For that, DNA sequences encoding universal T helper cell epitopes will be integrated into the vaccine. Furthermore, for effective loading of TAA- and T helper epitopes onto MHC-I and –II molecules, respectively, both lysosomal and proteasomal antigen presentation pathways will be induced by the DNA vaccine-dependent synthesis of the mutated ubiquitin with increased stability (polyubiquitination). An additional stimulation of APCs will be achieved by the vaccine-mediated synthesis of IL-15. After generation and characterization, antitumor efficacy of the new DNA vaccine will be evaluated in our preclinical NB model. For that, a new non-viral liposome-based nanoparticle vaccination strategy will be utilized. To overcome a tumor-mediated inhibition of immune response, an additional blockade of two immune checkpoints (PD-L1 + TIGIT) that have been shown to play an important role in cancer will be performed.In summary, we aim to generate a new generation cytokine-based DNA vaccine against different NB-specific TAAs that will be tested in combination with immune checkpoint blockade for induction of a long-lasting immune response against NB.